Specific Aim 1. Retrograde transport of late endosomes regulates autophagy-lysosomal function essential for neuron survival (Cai et al., Neuron 2010; Xie et al., Neuron 2015; Cheng et al., JCB 2015). Dynein motors drive retrograde transport of late endosomes, thus enhancing their trafficking from distal processes to the soma, where mature lysosomes are mainly localized. Our recent study uncovered a critical role for snapin in regulating late endocytic transport and endo-lysosomal trafficking. Snapin acts as a dynein motor adaptor by attaching dynein to late endosomes. The snapin KO neurons exhibit impaired retrograde transport of late endosomes, reduced lysosomal proteolysis, aberrant accumulation of immature lysosomes, and impaired clearance capacity of autolysosomes. Snapin deficiency leads to axonal degeneration and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes. Our studies elucidate a mechanism coordinating dynein-mediated late endocytic transport and lysosomal degradation capacity in neurons. Such a mechanism is critical for maintaining cellular homeostasis essential for neuronal survival. Autophagy-lysosomal dysfunction is one of the cellular defects contributing to the pathogenesis of major neurodegenerative diseases associated with accumulation of aggregation-prone proteins and damaged organelles. Specific Aim 2. Endo-lysosomal trafficking regulates presynaptic activity (Di Giovanni and Sheng, EMBO J 2015). Proper regulation of synaptic vesicle (SV) pool size is critical to maintain presynaptic activity. Because early endosomes represent crossroads between local SV recycling and endo-lysosomal degradation, this raises a fundamental question as whether SVs are sorted toward endo-lysosomal pathway for degradation. To address this, we applied snapin dominant-negative mutants combined with dual-channel time-lapse imaging in live cortical neurons. Our study reveals that dynein-driven late endosome transport regulates SV pool size. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin-/- phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endo-lysosomal pathway. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size through endosomal trafficking and sorting. Specific Aim 3. Mechanism removing autophagosomes from distal axons (Cheng et al., JCB 2015). Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process over a neurons lifetime. Newly formed autophagosomes are fused with late endosomes into amphisomes, or fused with lysosomes into autolysosomes for degradation. However, it is unknown how autophagosomes generated in distal axons acquire their retrograde motility toward the soma for degradation. We reveal a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Endosome-loaded dynein-snapin complexes mediate retrograde transport of autophagosomes upon their fusion into amphisomes in distal axons. This motor sharing mechanism enables neurons to maintain effective autophagic clearance, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular pathway to remove distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases. Specific Aim 4. Autophagy-lysosomal deficits contributes to ALS-linked early pathology (Xie et al., Neuron 2015). One pathological hallmark in fALS-linked motor neurons (MNs) is axonal accumulation of AVs, thus raising a fundamental question as to whether reduced autophagic clearance contributes to autophagic stress and axonal degeneration. We recently reveal progressive lysosomal deficits in spinal MNs beginning at early asymptomatic stages in fALS-linked mice expressing the human mutant SOD1G93A protein. Such deficits impair the degradation of AVs engulfing damaged mitochondria from distal axons. These early pathological changes are attributable to mutant hSOD1, which interferes with dynein-driven endo-lysosomal trafficking. Elucidation of this pathological mechanism is broadly relevant, because autophagy-lysosomal deficits are associated with several major neurodegenerative diseases. Therefore, enhancing lysosome function, rather than autophagy induction, is an alternative therapeutic strategy for ALS-linked clinical trials. Specific Aim 5. Synaptic cargo transport regulates synaptic maintenance and plasticity (Su et al., Nature Cell Biology 2004; Cai et al., Journal of Neuroscience 2007; Xiong et al., in preparation). The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity; this process requires the targeted delivery of newly synthesized synaptic cargoes from the soma to distal synapses. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes. Syntabulin-KIF5 coupling mediates axonal transport of synaptic components essential for presynaptic assembly and maintenance. Syntabulin loss-of-function impairs the assembly and maintenance of presynapses in developing neurons. Our recent study shows that syntabulin conditional KO mice display autism-like social interaction and impaired synaptic plasticity. Our studies establish that kinesin-mediated axonal transport is one mechanism underlying activity-dependent presynaptic plasticity. Selective Publications: Xiu-Tang Cheng, Bing Zhou, Mei-Yao Lin, Qian Cai, and Zu-Hang Sheng (2015). Axonal autophagosomes acquire dynein motors for retrograde transport through fusion with late endosomes. Journal of Cell Biology 209, 377-386. Xiu-Tang Cheng, Bing Zhou, Mei-Yao Lin, Qian Cai, and Zu-Hang Sheng (2015). Axonal autophagosomes use the ride-on service for retrograde transport toward the soma. Autophagy 11, 1434-1436. Jerome Di Giovanni and Zu-Hang Sheng. (2015) Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting. EMBO J 34, 2059-2077. Yuxiang Xie, Bing Zhou, Mei-Yao Lin, Shiwei Wang, Kevin D. Foust, and Zu-Hang Sheng. (2015) Endolysosome deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS-linked mice. Neuron 87, 355-370. Yuxiang Xie, Bing Zhou, Mei-Yao Lin, and Zu-Hang Sheng. (2015) Progressive endolysosomal deficits impair autophagic clearance beginning at early asymptomatic stages in fALS mice. Autophagy 11, 1934-1936. Sheng, Z.-H. (2017) The interplay of axonal energy homeostasis and mitochondrial trafficking and anchoring. (Invited review) Trends in Cell Biology 27, 403-416.